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. 2020 Mar 10;14(3):374–389. doi: 10.1016/j.stemcr.2020.02.004

Figure 6.

Figure 6

MERTK Mutations in Human RPE Abolish POS Ensheathment

(A) A schematic of the experiment in (B–H).

(B–D) Ten images containing around ten cells from each condition were analyzed. Significance was calculated using one-way ANOVA test. ns > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Data are represented as means ± SD. N = 3 biological repeats. In Figure S7 ensheathment rescue in isogenic control is shown. (B) Quantification of the number of sheets associated with POS per cell. (C) Quantification of the percentage of cells presenting sheets. (D) Quantification of the number of unensheathed F-POS per cell.

(E–H) SEM images of wild-type and MERTK mutant RPE treated with F-POS and 30% serum. Scale bar, 10 μm. (E) Wild-type hESC-RPE. (F) MERTK mutant MERTK-RPE. (G) MERTK mutant EX2-RPE. (H) MERTK mutant EX14-RPE.

(I) A schematic of the experiment in (J–W). Wild-type and MERTK mutant RPE (grown on transwells) were primed 1 h with 30% serum. Alternatively, wild-type RPE cells were primed with 5 μg/mL GAS6 or MFGE8 for 1 h. Next, cells were challenged with W-POS and incubated for 3 and 5 h at 37°C. Samples were next processed for SEM.

(J) Two types of W-POS particles were observed by SEM elongated (red arrow) and round-oval (blue arrow) outer segments. Scale bar, 5 μm.

(K) Whole POS particles are RHO rich. Scale bar, 3 μm.

(L–N) SEM images. Stages of POS binding, ensheathment, and ensheathment-mediated fragmentation. Scale bar, 5 μm. (L) Upon contact between hESC-RPE and W-POS in the presence of 30% serum, RPE extends its membrane sheets around the particle. (M) Next, RPE sheets invade POS and fragment them. (N) POS are completely fragmented before they are internalized.

(O) Analysis of W-POS ensheathment in SEM images at 3 h and fragmentation by 5 h in the presence of 30% serum. Data are represented as means ± SD. N = 3 biological repeats.

(P–W) Representative SEM images of the analysis shown in (O). Scale bars, 50 μm (P, R, T, and V). Zoomed-in SEM images of (Q), (S), (U), and (W). Scale bar, 10 μm. W-POS were ensheathed at 3 h (P and Q) and fragmented after 5 h by hESC-RPE treated with 30% serum (R and S), while EX2-RPE showed neither POS ensheathment at 3 h (T and U), nor ensheathment-mediated fragmentation at 5 h (V and W).